Whenever I revive any stock of RPMI 8226, clumps are being formed in the flasks. I revive my cells(5 X 10^6) in T-75 flask. I have pippetted them many times but they still form. Can we pass the cell suspension through a syringe to make a single cell suspension? Please suggest.
Dear Raman, I would first check if your cells are alive, simply by trypan blue exclusion. Clumps are sign of some kind of membrane damage and it is difficult to get rid off them. To avoid over-stressing the cells, I would pass the suspension trough a corning filter that fits in a 50 ml tube. In this way you will get rid of the clumps and allow only for the passage of single cells that you can replate in the flask. By the way, how do you freeze and thaw the cells?
Dear Danilo Sir, i have done that ,all the cells were alive except few cells. freezing protocol is like 5% DMSO, 10%FBS,in complete media. then cryo-vial is passed through a temp. gradient is formed begining with -20(3hrs), -80(o/n), then finally at -196'C.
thawing includes protocol given at atcc file attached.
Regards
Raman Kumar
Dear Raman,did the cells form clumps direct afer thawing or when they are a few passages in culture?
I would not put them through a syringe this would be a lot of stress for the cells. Maybe you could try to seperate them with Accutase?
Dear Sibylle, clumps can be seen in the passages next to the thawed one.. ..
Hi Raman, I suposse that the cells somehow have a problem with the freezing and thawing procedure, because you see the clumps in the passages next to thawed one.
The sugestion from Danilo sounds good, give it a try.
Regards
Sibylle
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